Anion-Sensitive, H<sup>+</sup>-Pumping ATPase of Oat Roots

Phosphorus is acquired by plant roots primarily via the high-affinity inorganic phosphate (P i ) transporters. The transcripts for P i transporters are highly inducible upon P i starvation, which also results in enhanced P i uptake when P i is resupplied. Using antibodies specific to one of the tomato P i transporters (encoded by LePT1), we show that an increase in the LePT1 transcript under P i starvation leads to a concurrent increase in the transporter protein, suggesting a transcriptional regulation for P i acquisition. LePT1 protein accumulates rapidly in tomato roots in response to P i starvation. The level of transporter protein accumulation depends on the P i concentration in the medium, and it is reversible upon resupply of P i . LePT1 protein accumulates all along the roots under P i starvation and is localized primarily in the plasma membranes. These results clearly demonstrate that plants increase their capacity for P i uptake during P i starvation by synthesis of additional transporter molecules.Phosphorus availability is considered one of the major factors that limits growth of plants in natural ecosystems. The concentration of available phosphorus is generally in the micromolar range, which is below that of many micronutrients (1). Consequently plants have developed several adaptive mechanisms to overcome P i deficiency. These include changes in root growth and architecture, increased production of phosphatases and RNases, altered activity of several enzymes of the glycolytic pathway (2, 3), and an increased P i uptake rate of roots (4). The ultimate consequences of these modifications are increased P i availability in the rhizosphere and enhanced uptake.Phosphorus is acquired by the plant roots in an energymediated cotransport process driven by a proton gradient generated by plasma membrane H ϩ -ATPases (5). The kinetic characterization of the P i -uptake system by whole plants and cultured cells indicates a high-affinity transport activity operating at low concentrations (micromolar range) and a lowaffinity activity operating at higher concentrations. The very low concentration (micromolar) of P i in soil solution suggests that high-affinity transporters are primarily involved in P i uptake by plants (3). The low-affinity system is apparently expressed constitutively, whereas the high-affinity system is induced under P i deficiency (6). The induction process appears to involve de novo protein synthesis, since inhibitors of protein synthesis drastically reduce the induction of high-affinity P i transport. The increased synthesis of a high-affinity carrier system has been proposed to be responsible for enhanced P i uptake observed under P i -deficiency conditions (6).High-affinity P i transporter genes have been cloned and characterized from fungi and from several plant species, including Arabidopsis, tomato, potato, Medicago, and Catharanthus (7). All the cloned P i transporters are integral membrane proteins containing 12 membrane-spanning regions, separated into two groups of 6 by a lar...

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“…17), tonoplast (NO 3 -sensitive ATPase, ref. 18), and mitochondrial membranes (oligomycin-sensitive ATPase, ref. 19).…”

Section: Methodsmentioning

confidence: 99%

Transcriptional regulation of plant phosphate transporters

Muchhal

Raghothama²

1999

Proc. Natl. Acad. Sci. U.S.A.

167

125

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“…Our results substantiate the suggestion inherent in the model that DA acts by direct interaction with the membrane. However, because DA disrupted ApH formation (Tables I and III) under conditions in which a A/if does not form (50 mm KCI) (6), it is evident that A/t was not required for DA action. Furthermore, the presence of Ai, did not enhance DA disruption of H+ electrochemical gradients (Table VI).…”

Section: Permeabilitymentioning

confidence: 99%

“…1 -3) were monitored in the presence of 50 mM KC1. Under these conditions, compensatory movement of Cl-, a permeant anion, during ApH development eliminates formation of A4l (6).…”

Section: Permeabilitymentioning

confidence: 99%

Diclofop-Methyl Increases the Proton Permeability of Isolated Oat-Root Tonoplast

Ratterman¹,

Balke²

1989

Plant Physiol.

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Diclofop-methyl (methyl ester of 2-[4-(2',4'-dichlorophenoxy)phenoxy]propionate; 100 micromolar) and diclofop (100 micromolar) inhibited both ATP-and PPi-dependent formation of H+ gradients by tonoplast vesicles isolated from oat (Avena sativa L., cv Dal) roots. Diclofop-methyl (1 micromolar) significantly reduced the steady-state H+ gradient generated in the presence of ATP. The ester (diclofop-methyl) was more inhibitory than the free acid (diclofop) at pH 7.4, but this relative activity was reversed at pH 5.7. Neither compound affected the rate of ATP or PPi hydrolysis by the proton-pumping enzymes. Diclofop-methyl (50, 100 micromolar), but not diclofop (100 micromolar), accelerated the decay of nonmetabolic H gradients established across vesicle membranes. Diclofop-methyl (100 micromolar) did not collapse K+ gradients across vesicle membranes. Both the (+)-and (-)-enantiomers of diclofop-methyl dissipated nonmetabolic H+ gradients established across vesicle membranes. Diclofopmethyl, but not diclofop (each 100 micromolar), accelerated the decay of H+ gradients imposed across liposomal membranes. These results show that diclofop-methyl causes a specific increase in the H permeability of tonoplast.The herbicide DM3 and its free acid form, DA, inhibit shoot and root growth and cause chlorosis and necrosis of oat and wild oat tissues (7,14,23,28). Ultrastructural damage observed in treated tissues includes breakdown of cellular membranes and destruction of cytoplasmic structure (5). Evidence indicates that they interfere with membrane-associated functions causing inhibition of mineral ion absorption (1), leakage of electrolytes from cells (7) cides is not known, presently two hypothetical mechanisms are being evaluated experimentally (10). In one model, the basis for structural and functional effects on cellular membranes is a direct interaction of the herbicide with the membrane or membrane components. A direct effect of DA on membrane function is indicated by the rapidity of depolarization of electrical potentials by DA (within 10 min [21,36]) and repolarization after removal of DA (40 s, with IAA addition [32]). The nature of the DA-membrane interaction or of the membrane components required for DA activity has not yet been determined. The second model suggests that membrane disruption is an indirect effect of herbicidal action resulting from an inhibition of the biosynthesis of membrane lipids. De novo fatty acid biosynthesis has been shown to be a sensitive site of action for DA (15,17). The I50 values for the inhibition by DA of ['4C]acetate incorporation by maize and oat chloroplasts were 0.2 and 0.1 M, respectively (15, 17). DA was more inhibitory than was DM (Iso = 10 uM) in these assays. Recent evidence indicates that the enzyme acetylCoA carboxylase is the site of inhibition by DA in this biosynthetic pathway ( 17).We have used an in vitro system of tonoplast vesicles isolated from oat root to study the possible direct action of DM and DA on membranes. With isolated membranes, effects of the h...

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“…In general, the purified H+ pump appeared to retain its native properties and conformation as shown by the striking similarity in sensitivity to several known inhibitors of the native tonoplast H+-ATPases coupled to H+ pumping. Chloride could stimulate H+ transport in native membrane vesicles either (a) by dissipation of the membrane potential by Cl-uptake via anion channels, (b) by direct activation of the ATPase, or (c) by both mechanisms (7). H+ transport of a crude reconstituted V-type ATPase from corn roots was previously shown to be dependent on Cl-(5).…”

Section: Purified H' Pump Was Inhibited By Bafilomycin Dccd and Nitmentioning

confidence: 99%

Proton Transport Activity of the Purified Vacuolar H⁺-ATPase from Oats

Ward

Sze

1992

Plant Physiol.

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To determine whether the detergent-solubilized and purified vacuolar H'-ATPase from plants was active in HW transport, we reconstituted the purified vacuolar ATPase from oat roots (Avena sativa var Lang). Triton-solubilized ATPase activity was purified by gel filtration and ion exchange chromatography. Incorporation of the vacuolar ATPase into liposomes formed from Escherichia coli phospholipids was accomplished by removing Triton X-100 with SM-2 Bio-beads. ATP of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. These results demonstrate conclusively that the purified vacuolar ATPase is a functional electrogenic H' pump and that a set of 10 polypeptides is sufficient for coupled ATP hydrolysis and H' translocation.An electrogenic H+-ATPase2 is located in the vacuolar membrane of higher plant cells (20 V-type H+-ATPases are large and multimeric and composed of a peripheral (cytoplasmic) sector containing the ATP hydrolytic site and a membrane integral sector that forms the proton transport pathway. Three major subunits are common to all V-type ATPases. The 70-and 60-kD subunits are present in three copies each in the peripheral sector (4), contain nucleotide-binding sites, and are thought to have catalytic and regulatory roles, respectively. The 16-kD subunit is a proteolipid that binds DCCD (an inhibitor of V-type ATPases) and is present in six copies in the integral sector (4, 12). The plant V-type H+-ATPases purified from either red beet storage tissue (17), mung bean hypocotyl (13), barley roots (9), or oat roots (18, 24) contain 9 or 10 subunits. Hence, the plant V-type H+-ATPase is similar in subunit complexity to those of other eukaryotic sources (10).The purified vacuolar H+-ATPase (650 kD) from oat roots contains 10 subunits, with molecular masses of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kD (24). Six of these constitute the large peripheral sector, and the remaining four polypeptides of 32, 16, 13, and 12 kD make up the integral sector. The large peripheral sector is visible as a knob and stalk structure on the surface of tonoplast vesicles (24). The peripheral complex of the enzyme can be dissociated from the integral portion by chaotropic anions, such as I-and NO3-, which decrease both ATP hydrolytic and H+ transport activities. However, we have recently shown that the dissociated and inactive H+-ATPase from oat can be reassembled to produce a fully functional H+-translocating ATPase (22).Although V-type H+-ATPases from several plant sources have been purified, H+ transport of a purified plant H+-ATPase has not been previously demonstrated. To test whether the purified H+-ATPase from oats retained H+ transport activity and to study the regulation of the H+-ATPase 925 www.plantphysiol.org on May 11, 2018 -Published by Downloaded from

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Anion-Sensitive, H⁺-Pumping ATPase of Oat Roots

Cited by 113 publications

References 36 publications

Transcriptional regulation of plant phosphate transporters

Transcriptional regulation of plant phosphate transporters

Diclofop-Methyl Increases the Proton Permeability of Isolated Oat-Root Tonoplast

Proton Transport Activity of the Purified Vacuolar H⁺-ATPase from Oats

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Anion-Sensitive, H+-Pumping ATPase of Oat Roots

Cited by 113 publications

References 36 publications

Transcriptional regulation of plant phosphate transporters

Transcriptional regulation of plant phosphate transporters

Diclofop-Methyl Increases the Proton Permeability of Isolated Oat-Root Tonoplast

Proton Transport Activity of the Purified Vacuolar H+-ATPase from Oats

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Product

Resources

About

Anion-Sensitive, H⁺-Pumping ATPase of Oat Roots

Proton Transport Activity of the Purified Vacuolar H⁺-ATPase from Oats